pH和氮素对球等鞭金藻胞外碳酸酐酶活性的影响

采用pH电极法研究pH和氮素对球等鞭金藻胞外碳酸酐酶活性的影响．结果表明,球等鞭金藻具有基本的胞外碳酸酐酶活性,且pH和氮素均能明显地诱导其胞外碳酸酐酶活性．pH从6．5升高到9．0时,球等鞭金藻胞外碳酸酐酶活性明显增加,pH8．5时酶活性达到最大．球等鞭金藻的pH补偿点为9．86,表明其具有利用HC03-的能力．氮素对胞外碳酸酐酶活性诱导也具有重要的影响,3种氮素NH4Cl、NaNOM3、CO（NH2）2在2．0mmoL／L时诱导的酶活性均明显高于8．5mmoL／L时的诱导的酶活性,且有机氮诱导的酶活性明显大于无机氮诱导的酶活性,硝态氮诱导的酶活性大于氨态氮诱导的酶活性．     

Effect of acetazolamide on stable carbon isotope fractionation in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> and <Emphasis Type="Italic">Chlorella vulgaris</Emphasis>

The effect of extracellular carbonic anhydrase (CAex) on stable carbon isotope fractionation in algae is still unclear. The stable carbon isotope composition and algal growth in the presence and absence of the membrane-impermeable CA inhibitor acetazolamide were compared in Chlamydomonas reinhardtii and Chlorella vulgaris. The CAex of both algal species contributed about 9‰ of the stable carbon isotope fractionation and exhibited a dosage effect. Therefore, evidence in vivo that CAex leads to a larger carbon isotope fractionation of algae is presented.     

碳酸酐酶在Fe3O4表面固定及性能分析研究

首先将羧基官能团引入纳米级Fe_3O_4表面,并用交联剂戊二醛将碳酸酐酶(carbonic anhydrase,CA)固定到Fe_3O_4上,即得到固定化酶。采用比表面积(BET)、热重分析(TG)、傅里叶红外(FTIR)、X射线衍射(XRD),扫面电镜(SEM)等手段对载体材料和固定化酶的物理化学性质进行表征对比。以对硝基苯酚乙酸酯(p-NPA)为底物,采用紫外分光光度计测定酶活。结果表明,制备的载体材料具有较大的比表面积,表面羧基化后,载体共价结合后的有机物含量达到11.24%;载体材料表面不存在C=C。采用戊二醛进行酶的固定化试验时,固定化酶活性最大可达到68.26%;且固定化酶比游离态酶具有更高的操作稳定性和贮藏稳定性。本方法有望将固定化酶应用在连续式填料塔反应器中以提高其低浓度CO_2的去除效率。     

Photosynthetic bicarbonate utilization inPorphyra haitanensis (Bangiales, Rhodophyta)

The activities of carbonic anhydrase (CA) and photosynthesis of Porphyra haitanensis were investigated in order to see its photosynthetic utilization of inorganic carbon source. Both intra- and extra-cellular CA activities existed in the thallus. CA inhibitors, acetazolamide (AZ) and ethoxyzolamide (EZ), remarkably depressed the photosynthetic oxygen evolution in seawater of pH 8.2 and 10.0, and EZ showed stronger inhibition than AZ. The observed net photosynthetic rate in seawater of pH 8.2 was much higher than that of CO2 supply theoretically derived from spontaneous dehydration of HCO3-. P. haitanensis also showed a rather high pH compensation point (9.9). The results demonstrated that P. haitanensis could utilize bicarbonate as the external inorganic carbon source for photosynthesis. The bicarbonate utilization was closely associated with dehydration catalyzed by extracellular CA activity. The inorganic carbon composition in seawater could well saturate the photosynthesis of P. haitanensis. The low Km value and compensation points for inorganic carbon reflected the existence of CO2- concentrating mechanism in this alga.     

磁场对碳酸酐酶的影响

建立了流动注射分析方法 ,评价了碳酸酐酶 (CA)的活性 ,并用此方法研究了磁场对酶催化活性的影响。发现磁场作用使酶活力升高 ,2 10mT下作用 4h ,其活力增加了 17% ;随着磁化时间的延长 ,其磁场效应基本上趋于一致 ,且观察到了磁酶效应的可逆性。实验还发现磁场使酶 -底物反应的活化能降低 ,并对可能机理作了初步探讨。     

高浓度CO_2对小新月菱形藻胞外碳酸酐酶活性和光合作用的影响

以赤潮硅藻小新月菱形藻(Nitzschia closterium var.minutissima)为实验材料,研究了短期内(12 h)高浓度CO2(5%CO2)对其胞外碳酸酐酶活性和光合作用的影响,结果显示,高浓度CO2培养导致小新月菱形藻胞外碳酸酐酶活性、叶绿素a和叶绿素c含量明显下降.与通空气培养(0.035%CO2)相比,在短期内(12 h)胞外碳酸酐酶活性下降了75.4%,叶绿素a、c含量分别降低了5.6%和7.3%;高浓度CO2培养下最大光化学效率(Fv/Fm)、实际光化学效率(Yield)和光化学淬灭系数(qP)下降,但非光化学淬灭系数(gN)升高.研究结果表明,高浓度CO2对胞外碳酸酐酶活性具有明显的抑制作用,小新月菱形藻通过调整光系统Ⅱ的能量流动和能量利用效率以适应高浓度CO2的环境.     

Relationship among Photosys-tem Ⅱ carbonic anhydrase,extrinsic polypeptides and manganese cluster

Effects of Photosystem Ⅱ (PS Ⅱ) extrinsic polypeptides of oxygen-evolving complex and manganese clusters on PSⅡ carbonic anhydrase (CA) were studied with spinach PSⅡ membranes. The result supported that membrane-bound CA is located in the donor side of PSⅡ. The extrinsic polypeptides played an important role of maintaining CA activity. After removing manganese clusters, oxygen evolution activity was inhibited, but PSⅡ-CA activity was unchanged. It was concluded that CA activity is independent of the presence of manganese clusters, and was not directly correlated with oxygen evolution activity.     

Models for metal ion function in carbonic anhydrase.

A model catalyst is described which has properties in common with carbonic anhydrase. The model demonstrates the availability of a mechanism, previously only hypothetical, for the action of the enzyme. It also shows, however, that this mechanism alone is not adequate to produce the high activity of the enzyme.     

虹鳟假鳃组织中的碳酸酐酶免疫细胞化学定位

研究目的：假腮的功能早已引起科学家兴趣,但还有待阐明。本文通过研究硬骨鱼类品种虹鳟鱼（Oncorhynchusmykiss）的假腮碳酸酐酶的免疫定位,来探讨假腮碳酸酐酶的生理功能。研究方法：免疫组织化学染色技术。重要结论：免疫组化结果显示碳酸酐酶分布在假腮细胞中,更精确地说是在其细胞顶端分布。细胞基底端、管状系统、毛细血管和柱细胞均无免疫染色。免疫细胞化学定位进一步验证了这些结果,并显示一部分是细胞质碳酸酐酶,其余的与细胞膜结构连接。此外,腔隙层未显示出免疫过氧化物酶的活性。本研究揭示了假腮碳酸酐酶的功能与细胞外介质有关,碳酸酐酶能干预传入神经纤维刺激机制。     

Exogenous carbon acquisition of photosynthesis in Porphyra haitanensis (Bangiales, Rhodophyta) under emersed state

The photosynthetic performances of Porphyra haitanensis thalli were investigated in order to understand its mechanisms for exogenous carbon acquisition during emersion at low tide. The emersed photosynthesis was studied by altering the pH value in the water film on the thalli surface, treating them with carbonic anhydarase inhibitors (acetazolamide and 6-ethoxyzolamide), adjusting the CO2 concentrations in the air, and comparing the theoretical maximum CO2 supply rates within the adherent water film with the observed photosynthetic CO2 uptake rates. It was found that the principal exogenous inorganic carbon source for the photosynthesis of P. haitanensis during emersion was atmospheric CO2. The driving force of CO2 flux across the water film was the CO2 concentration gradient within it. Carbonic anhydrase accelerated both extracellular and intracellular CO2 transport. The emersed photosynthesis of P. haitanensis was limited by the present atmospheric CO2 level, and would be enhanced by atmospheric CO2 rise that would trigger global warming.     

碳酸酐酶在四氧化三铁上固定化及其性能表征研究

以四氧化三铁为载体,利用共价结合和交联法相结合的方法制备固定化碳酸酐酶(CA),通过单因素试验,得出CA最佳固定化条件,并对固定化酶进行性能表征.结果表明,在最优的固定化条件下,固定化酶的酶活回收率可达到66.90%,且其pH值稳定性、操作稳定性、热稳定性和贮藏稳定性明显高于同等条件下的游离酶,该固定化酶经5次催化水解底物后仍能保持62.58%的相对酶活.     

发光二极管作为组培光源的特性分析与应用

为实现组织培养光源的有效调控，节约组培光照耗能，同时为了解发光二极管（LED）光源对组培苗生长的影响，从LED的光电、衰减、透光特性以及有效发光效能等几个方面对LED作为组培光源进行了特性分析．研究结果表明：LED光源透光性强、衰减有规律，它的光电特性具有良好的线性关系，有效发光效能也明显高于普通日光灯，红、白光LED光源通过电能转化的有效光能达到了日光灯的3倍．同时，通过对光源效果的试验研究，结果表明，蓝光LED光源处理的诸葛菜和油菜组培苗鲜重增量百分数最大，分别达到了595％和490％，碳酸酐酶活性最强，分别达到了227和182WAU g^-1FW．研究结果将为LED作为组培光源的控制提供理论依据．     

Determination of sulfonamides with fluorescent inclusion complex of carbonic anhydrase and dansylamide

A new fluorimetric method for determination of sulfonamides was described based on the formation of a fluorescent inclusion complex of carbonic anhydrase(CA) with dansylamide(DNSA). The binding of DNSA to CA resulted in an enhancement in the fluorescence emission at 460 nm with excitation at 280 nm. Dissociation constants were determined for the carbonic anhydrase sulfonamide complexes. Linear calibration graphs of sulfonamides were obtained within a concentration range of 0- 0.058 μg/mL for DNSA; 0-0.344 μg/mL for sulfanilamide (SAN) and 0-0.286 μg/mL for P toluenesulfonamide (PTSN). The relative standard deviations were within 1.8%-4.2%. Limits of detection for DNSA, SAN and PTSN were 0.84, 19.5 and 6.1 ng/mL, respectively. The method was applied to the determination of sulfonamides in cow milk at ng/mL level.     

Thermodynamic study on conformation change of carbonic anhydrase

The effect of pHs on structural stability of the zinc-containing metalloenzyme carbonic anhydrase has been studied by means of differential scanning calorimetry. The rule of the conformational change for the enzyme with pH change has been found by thermodynamic analysis. The experimental results also provide valuable information: in the pH range from 5.26 to 9.04, two independent endothermal peaks were observed on DSC thermogram. It shows the existence of two structural domains in the molecule. The transition temperature and enthalpy of the two domains are different.     

Structure and metal exchange in the cadmium carbonic anhydrase of marine diatoms

Carbonic anhydrase, a zinc enzyme found in organisms from all kingdoms, catalyses the reversible hydration of carbon dioxide and is used for inorganic carbon acquisition by phytoplankton. In the oceans, where zinc is nearly depleted, diatoms use cadmium as a catalytic metal atom in cadmium carbonic anhydrase (CDCA). Here we report the crystal structures of CDCA in four distinct forms: cadmium-bound, zinc-bound, metal-free and acetate-bound. Despite lack of sequence homology, CDCA is a structural mimic of a functional beta-carbonic anhydrase dimer, with striking similarity in the spatial organization of the active site residues. CDCA readily exchanges cadmium and zinc at its active site--an apparently unique adaptation to oceanic life that is explained by a stable opening of the metal coordinating site in the absence of metal. Given the central role of diatoms in exporting carbon to the deep sea, their use of cadmium in an enzyme critical for carbon acquisition establishes a remarkable link between the global cycles of cadmium and carbon.     

基于Scatchard法的碳酸酐酶-脂质体与药物结合常数测定

 为更好地模拟药物小分子与碳酸酐酶在体内的相互作用,采用碳酸酐酶-脂质体毛细管电泳法,以4-羧基苯磺酰胺为分析对象建立碳酸酐酶药物筛选模型,并以此模型为基础计算12 种药物小分子与碳酸酐酶的结合常数,分析药物小分子与碳酸酐酶的相互作用。结果表明,4-羧基苯磺酰胺与碳酸酐酶-脂质体的结合常数为1.172×10⁴ mL·g^-1,具有较强的相互作用。基于此,筛选出4 种(咖啡酸、L-抗坏血酸、2, 4-二氯-5-磺酰胺基苯甲酸和4-氯-3-磺酰胺基苯甲酸)和5种(阿魏酸、马兜铃酸、没食子酸、原儿茶酸、烟酸)与复合物具有较强或较弱相互作用的药物。通过该法可快速、有效、经济地测定碳酸酐酶或其他靶标与药物的相互作用,缩短药物研发周期。     

锌对青花菜叶片光合特性的影响

采用砂培的方法,利用气体交换测定技术,研究适量施锌对青花菜叶片光合特性的影响,揭示锌提高青花菜光合能力的生理机制.结果表明:适量施锌较不施锌叶片的叶绿素a和叶绿素b质量分数分别提高8.72%,10.17%,碳酸酐酶(CA)活性提高152.94%;净光合速率(Pn)提高14.32%,气孔导度(Gs)增大41.55%,胞间CO2浓度(Gi)增加26.17%;表观量子效率(AQY)提高12.14%,羧化效率(CE)提高7.59%.锌通过增大叶片的气孔导度,增加胞间CO2浓度,降低气孔限制来提高净光合速率;通过提高CA活性来提高叶片的羧化能力和光合能力.     

Biochemistry: a cadmium enzyme from a marine diatom

The ocean biota contains a vast reservoir of genomic diversity. Here we present the sequence and preliminary characterization of a protein that is a cadmium-containing carbonic anhydrase from the marine diatom Thalassiosira weissflogii. The existence of a cadmium enzyme in marine phytoplankton may indicate that there is a unique selection pressure for metalloenzymes in the marine environment, and our discovery provides a long-awaited explanation for the nutrient-like behaviour of cadmium in the oceans.     

Enhanced urease inhibition activity of Ag nanomaterials capped with N-substituted methyl 5-acetamido-β-resorcylate

Medically, bacterial ureases are important virulent factors and are used for treatment of peptic ulcers and urinary tones. Reported urease inhibitors are associated with various side effects including antibiotic resistance as a major one. Still there is an urgent need to synthesize new urease inhibitors. In this context we have synthesized new urease inhibitor i.e. AgL that is composed of Ag nanomaterials capped with N-substituted methyl 5-acetamido-β-resorcylate(L). The conjugation of L to silver was confirmed through FTIR, UV–vis and TEM analysis.Bare silver nanomaterials(Ag) were also prepared. The stability of AgL nanostructures was determined against various parameters(temperature, high salt concentration, pH) and found to be stable. The in vitro antimicrobial(antibacterial, antifungal), enzyme inhibition(xanthine oxidase, urease, carbonic anhydrase, α-chymotrypsin,cholinesterase) and antioxidant activities of AgL were investigated and compared with Ag, L and standard drugs.In comparison to other bioactivities, AgL shows statistically enhanced selective enzyme inhibition activity against urease enzyme. Urease inhibition activity of AgL was significantly greater than standard drug(thiourea),L and Ag. On a per weight basis, AgL required about 11–18 times less amount of L for inhibition of urease enzyme.     

稀土对浮萍引起水体富营养化的光合作用影响机理初探

为探究外源稀土化合物对水生植物引起水体富营养化的影响机理,选取紫背浮萍作为研究对象,设置受控实验,采用生物化学及荧光光谱技术分析稀土铈化合物对浮萍生长状况及其光合作用的影响进行研究.实验结果显示:适宜浓度范围内的稀土铈能有效促进浮萍对N、P的吸收,提高碳酸酐酶活性(最大值为0.684 EU/μg),并能提高光能利用和转化效率,光合作用得到增强,从而促进植物生长,浮萍生长率最高可达到63.85%.进而,随着浮萍繁殖密度的增加,水体DO值降低,而COD值上升(最大值为6.448 mg/L),最终导致水质逐渐恶化.研究结果表明,适量稀土对浮萍类小型水生植物具有生长促进作用,对水体富营养化进程发展起到正催化作用.这无疑为稀土可持续开发和利用提供了基础理论,为研究稀土对水体富营养化的促进规律和水域污染综合防治提供了科学依据.